Split-YFP-coupled interaction-dependent TurboID identifies new functions of basal cell polarity in <i>Arabidopsis</i>.

Abstract

The formation of a body axis is one of the fundamental steps in developmental patterning in multicellular organisms. Ectopic expression of the stomatal protein BASL (BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE) reveals a proximal-distal cell polarity field in the leaf and an apical-basal field in the hypocotyl and root of <i>Arabidopsis</i>. This provides a framework for uncovering molecular components of body-axis cell polarity in higher plants. In this study, we developed a proximity labeling-based complementation system, termed tSYID (<u>t</u>andem fusion of <u>S</u>plit-<u>Y</u>FP and Turbo<u>ID</u>), to enable simultaneous visualization and identification of the tissue-wide cell polarity module marked by interacting BASL and BREVIS RADIX-LIKE 2 (BRXL2) proteins in <i>Arabidopsis</i>. This contact-based tSYID system focuses on identifying the protein interactome proximal to the protein-protein interaction site. By combining proximity labeling and mass spectrometry, we identified protein candidates associated with the tSYID-BASL/BRXL2 module, many of which remain uncharacterized in plants. Among them, an AGC protein kinase displayed highly polarized localization at the basal membrane in the hypocotyl and root. Knocking out two close <i>AGC</i> homologs in <i>Arabidopsis</i> resulted in compromised hypocotyl growth in the dark, suggesting a functional connection between basal cell polarity and plant upward growth. Our study demonstrates the power of using the Split-TurboID strategy to uncover proximal proteomes near protein activity sites and highlights an underappreciated crosstalk between developmentally programmed body axes and environmentally influenced growth vectors.